A Novel Multiplex RT-PCR Assay For The Detection Of Chromosomal Translocations Of Leukemia

Objective: The study was to develop a novel multiplex RT-PCR method mediated by tag primers to detect common recurrent chromosomal translocations in leukemia rapidly and accurately.Methods: Primers of this thesis include tag primers, specific primers and tag-specific primers.(1) Tag primers were firstly designed from some genomes of bacterium or virus with the criteria of no homologous sequences to human genome. Tag primers were then tested using human genomic DNA and c DNA. Only those that had no amplified products with human DNA or c DNA templates were kept for further applications. Selecting the recurrent transcriptions in leukemia case according to the leukemia database. After primers specific for each fusion gene were designed, Tag A1 primer and Tag A2 primer were added to all reverse primers and forward primers. The tag template was prepared by PCR amplification. After amplifying GAPDH gene with tag-specific primers, the 5’ ends and 3’ends of PCR products were added with Tag A1 primer and Tag A2 primer.(2) The mutant templates were obtained by overlap extension PCR(SOE-PCR). All the final PCR products were gel purified and then subcloned into the PGM-T vector. PCR on serial dilutions of cloned transcripts were used to assess the specificity and sensitivity of the primers.(3) To establish a multiplex PCR for fusion genes, two sets of primers specific for each fusion gene and two control genes were designed. Six mutant type templates were obtained by SOE-PCR technology: BCR/ABL e1a2, BCR/ABL e13a2, PML/RARα type L, PML/RARαtype S, AML1/ETO, E2A/PBX1 type 1. These templates were used in the detection of the first multiplex assay. In addition to the six templates of the first assay, the second multiplex assay was also targeted at E2A/HLF, TEL/AML1, TEL/ABL, CBFβ/MYH11 type A and SIL/TAL1. These plasmids were extracted and confirmed by direct sequencing. PCR on serial dilutions of cloned transcripts were used to assess the specificity and sensitivity of these multiplex assays. The primers and reaction conditions were optimized for the specificity and sensitivity of the assays. Detection of the four fusion genes consists of atwo-step PCR. A set of specific primers with added tag primers for each fusion gene was used in the first step. Tag A1 primer and Tag A2 primer were used in the second step.These two assays were applied in the diagnosis of blood samples collected from healthy individuals and patients.Results: Two multiplex RT-PCR systems in this study can specifically detect positive plasmids with known transcripts. The first system aimed at four fusion genes including BCR/ABL, AML1/ETO, PML/RARα and E2A/PBX1. The sensitivity of this system was up to 10 copies. The second system aimed at nine fusion genes including BCR/ABL,AML1/ETO, PML/RARα, E2A/PBX1, E2A/HLF, TEL/AML1, TEL/ABL, CBFβ/MYH11 and SIL/TAL1. The sensitivity was up to 100 copies. In the diagnosis of clinical specimens, all the chromosomal translocations were detected.Conclusion: This newly developed multiplex RT-PCR system can be used to detect the selected recurrent chromosomal translocations specifically and sensitively. It is novel,accurate, time and cost effective in the detection of leukemia-specific chromosomal translocations. Assays with this new strategy will have a potential in detecting more chromosomal translocations in leukemia.